Primary Dengue and Long-Term Health Status in Madeira Island, Portugal: A Retrospective Questionnaire-Based Study

ABSTRACT. Dengue is among the most important mosquito-borne viral diseases worldwide. Although its acute manifestations are well known, little is known about the long-term impact of dengue on the population’s health status. Madeira Island experienced a single outbreak of autochthonous dengue from September 2012 to March 2013. To extend our knowledge about the clinical impact of the outbreak on this naive population, we applied an online questionnaire to 168 adults diagnosed with dengue at the time to characterize retrospectively their symptoms during the infection and to identify long-term manifestations, possibly triggered by dengue. The most frequent symptoms during the clinical period, reported by more than three-quarters of our participants, were fever, myalgia, extreme tiredness, and headaches, whereas vomiting, pruritus, nausea, retro-orbital pain, and arthralgia occurred in 35% to 50% of participants. In the 8 years after dengue, 61.5% of participants reported at least one recurrent previously nonexistent symptom, the most frequent being headaches, abundant hair loss, extreme tiredness, arthralgia, and myalgia, experienced by 25% to 35% of participants. Nearly 20% of the participants with persistent symptoms reported the onset of chronic illness in the 4 years after dengue, most frequently ophthalmological and autoimmune diseases (5.6% each), versus only 2.2% of chronic disease onset in participants without persistent symptoms. Our results suggest that the occurrence of persistent symptoms after primary dengue might be more frequent than anticipated and may persist for several years, having an impact on the health status and well-being of a considerable proportion of the infected population.


INTRODUCTION
Dengue, a vector-borne disease caused by one of the four known serotypes of dengue virus (DENV), has become endemic in many tropical and subtropical regions, 1 where it represents a major health concern.During the past 5 decades, the incidence of dengue epidemics has increased 30-fold, reaching nowadays 390 million annual infections, with new countries being affected. 2][6][7][8] To gain a better understanding of the real impact of a dengue outbreak on the health status of a population, it is crucial to evaluate the clinical manifestations during active infection but also those that become more evident or appear after the recovery phase and persist chronically.Despite the recognition of late complications and unusual manifestations by the WHO, 9 little is known about the long-term consequences of dengue.1][12] A few studies have published evidence of an even longer duration of persistent symptoms, up to 2 years after infection. 6,13,14Additionally, long-term manifestations such as liver injury, 15 cardiovascular sequelae (reviewed in Rahim et al.), 16 and neurologic and ocular complications (reviewed in Trivedi and Chakravarty) 17 have also been described.Infection with DENV can also favor Guillain-Barr e or chronic fatigue syndromes, 17,18 as observed in other viral infections. 19,20he only outbreak of autochthonous dengue in Madeira Island, Portugal, was caused by DENV1 serotype and occurred between September 2012 and March 2013, leading to more than 2,000 dengue cases assisted in public healthcare, without severe clinical forms or fatalities. 21The clinical picture when seeking medical assistance, which generally happened during the febrile period, may have been underestimated because some of the dengue manifestations only appear later on (e.g., petechiae) or may be temporary (e.g., rash). 22Additionally, many of the patients did not qualify for follow-up or further clinical surveillance, and thus no registry of expanded manifestations exists beyond the acute phase of the infection.
To deepen our knowledge about the clinical impact of the Madeira 2012 dengue outbreak, we applied an online questionnaire to dengue cases diagnosed at the time, aiming at 1) a retrospective characterization of their symptoms during the entire infection period, from the early onset of symptoms to the convalescence phase, and 2) an assessment of their health status before and after dengue, identifying long-term manifestations possibly triggered by dengue.We expected that the clinical manifestations reported here are a fair representation of DENV1 infection in a naive population, not biased by the protective or facilitating immune response established against previous infections.

MATERIALS AND METHODS
Ethics statement.The study protocol was approved by the Ethics Committee for Health of the regional healthcare services of "Servic¸o de Sa ude da Região Aut onoma da Madeira (RAM), Entidade P ublica Empresarial da RAM" (SESARAM EPERAM) (approval no.49/2021).All participants gave their informed consent before starting the online questionnaire.All procedures complied with the Declaration of Helsinki of the World Medical Association and regulations for the protection of human subjects, rights, and personal data of the enrolled participants.
Selection of participants and questionnaire design.In 2021, we conducted a retrospective questionnaire-based study applied to patients with clinical and/or laboratory diagnoses of dengue fever in 2012-2013 by the regional healthcare services of SESARAM EPERAM, according to WHO diagnostic criteria 4 and following the guidance of the European Center for Disease Control (ECDC) experts assigned to the Madeira outbreak.According to the protocol established at SESARAM EPERAM during the outbreak, the laboratory diagnosis of dengue was done using different commercial rapid tests for DENV NS1 antigen detection (further details are available in the ECDC 2014 report). 21Additionally, reverse transcription polymerase chain reaction was performed for specimens from patients within 7 days from the onset of fever, and IgM and IgG capture ELISA was used for testing patients with fever onset longer than 7 days. 21The invited participants were aged between 16 and 62 years at the time of the outbreak.
The questionnaire was newly designed by the authors as a standardized form to collect directly observable parameters, that is, the clinical signs and symptoms experienced during DENV infection and subsequent persistent symptoms or possible sequelae.The multiple-choice options regarding symptoms during the infection and persistent symptoms were based on 1) the more commonly reported clinical parameters in the literature; 2) the findings of Garc ıa et al., 14 to our knowledge the only study reporting long-term persistent symptoms, 2 years postinfection; and 3) the clinical information obtained through a face-to-face questionnaire previously carried out as part of recruitment for a serological study targeting the same population as in the current study.In all questions, an open-field option allowed participants to report other information not included in the multiple options.The questionnaire's structure, content, and applicability to the studied population were evaluated by a panel of epidemiologists from the Faculty of Medicine of the University of Lisbon and by the SESARAM Internist member of this research work, who was involved in the management of the dengue outbreak of 2012-2013.Questionnaire reliability was determined using Cronbach's alpha test, and the estimated value for overall items (alpha 5 0.843) indicated its internal consistency.For the purposes of this study, the questionnaire was structured into three main sections (Figure 1): Section 1 was intended to characterize the signs/symptoms during the infection; Section 2 aimed at evaluating the general health status, before and after the infection, to infer about persistent manifestations after dengue; and Section 3 assessed demographic data of the participants.

STATISTICAL ANALYSES
The questionnaire was uploaded to the online SurveyMonkey V R platform (Momentive Inc., San Mateo, CA), an open-source and cloud-based platform that allows mobile data collection and complies with strict privacy and safety policies.An invitation with a link specifically generated for this questionnaire was sent to the mobile contacts of eligible participants, allowing them to respond anonymously and only once.Completed surveys were reviewed for data entry errors by the senior project members.
Data were extracted in .xlsformat and further edited for analyses in SPSS, version 28.0.0.0 (SPSS Inc., Chicago, IL).For each variable, valid participants were considered as those selecting "yes" or "no," and participants indicating "I don't know/won't answer" or leaving blank fields were excluded.The independence of categorical variables, overall and stratified by sex, was tested by univariate analyses using Pearson's x 2 tests (or Fisher's exact tests, when at least one of the expected values was ,5).Odds ratio (OR) and FIGURE 1. Flow diagram of the sections of the questionnaire and the number of valid participants per subject.* The number of valid participants who responded to each symptom in this question is variable because of the number of responders selecting "I don't know/I don't remember" for each symptom.respective 95% CI were calculated only for significant associations.Mean age differences, overall and per sex, were assessed by Mann-Whitney (or t-tests, according to the results of Shapiro-Wilk tests).Logistic regression was used to identify predictive variables for symptom persistence.All results were interpreted at a significance level of 0.05.

RESULTS
Characterization of participants.The invitation for participation in the survey was sent to 952 contacts (40.2% male, 59.8% female; aged 23-70 years, mean age 46.0 6 13.4 years (equivalent to a mean age of 38.0 years at the time of the outbreak), ascertained from the SESARAM EPERAM database as having had clinical and/or laboratory diagnosis of dengue fever during the outbreak.Of the 178 participants, not all were considered valid: a few participants did not answer the survey sections referring to signs/symptoms at the time of the infection (n 5 6), to the general health status before the infection (n 5 6), and/or to recurrent signs and symptoms after the infection (n 5 10), which were considered relevant for the purpose of our study and were therefore excluded from the analyses.Thus, 168 individuals, most of Portuguese nationality (98.1%), were considered valid participants.In terms of sex (32.7% male, 67.3% female) and age range (aged 24-70 years; mean age 43.9 6 11.9 years, equivalent to a mean age of 35.9 years at the time of the outbreak) (Figure 2), this is a representative subset of the invited participants because no significant differences were found when testing for proportions and means (both P-values .0.05).
When comparing the age distribution of participants exhibiting or not exhibiting each symptom, significant differences were found only for arm and leg swelling, which was more common in older participants (40.1 6 12.0 years versus 35.1 6 11.8 years, P 5 0.037; Table 1).No significant differences were observed for the mean age of males and females presenting with each symptom (Table 1).
Health status before dengue.Participants were asked about the existence of chronic diseases before dengue in 2012-2013.Most (72.6%, n 5 122) affirmed not having a diagnosis of chronic diseases at that time, whereas 27.4% (n 5 46) had one or more chronic diseases (Table 2).The most frequently reported comorbidities by our participants were hypertension (n 5 16), diabetes (n 5 7), and respiratory disease (n 5 5).It was also noted that, as expected, the mean age of participants with chronic diseases (41.2 6 11.4 years) was significantly higher than those without chronic diseases (34.1 6 11.6 years; P ,0.001).Also noteworthy, the mean age of participants with respiratory and autoimmune diseases was much lower (,30 years) than the mean age for other chronic diseases (over 44 years).Hypertension was the most reported chronic disease, and therefore we tested the association of this condition with each symptom during dengue by comparing their frequencies in hypertensive and nonhypertensive participants.For these comparisons, hypertension was suggested as a risk factor for arm and leg swelling (P 5 0.025, OR 5 3.743, 95% CI: 1.231-11.380)but protective against retro-orbital pain (P 5 0.029, OR 5 0.256, 95% CI: 0.070-0.936;data not shown).
Long-term persistence of clinical symptoms after dengue.In Section 2 of the questionnaire, participants were asked about persistent symptoms after dengue, among those symptoms shown in Table 3.To distinguish between occasional symptoms of unspecific cause and recurrent clinical manifestations that could point to sequelae or chronic health problems, we centered our attention on previously nonexistent or rare symptoms that became frequently reported (several times per year) after dengue.Considering that previous chronic diseases may influence the type and frequency of post-dengue recurrent symptoms, participants reporting comorbidities with onset before 2012-2013 were excluded from this analysis.Note the lower response rate in this and the following sections, with dropout of nine participants (four with previous chronic disease and five without previous chronic disease; Figure 1).
Additionally, female sex was suggested as a risk factor for the persistence of at least one symptom in our dataset because these were reported by 72.6% of females compared with 51.0% of males (P 5 0.012, OR 5 2.553, 95% CI: 1.273-5.118).On the other hand, the mean age of participants did not seem to influence the long-term persistence of at least one symptom.When assessing the frequencies of each recurrent symptom according to sex, we found significant differences in headaches (P 5 0.028, OR 5 2.847, 95% CI: 1.099-7.377)and abundant hair loss (P 5 0.019, OR 5 3.409, 95% CI: 1.180-9.851),with a higher frequency in females (Table 3).Additionally, the mean age of participants was found to correlate with persistent headaches (P 5 0.029), which were more common in younger patients, whereas arthralgia (P ,0.001) and bone pain (P ,0.001) prevailed in older patients (Table 3).
When testing the association of symptoms at the time of active infection as prognostic factors for the long-term persistence of at least one symptom, we found by univariate analyses that breathing difficulties were associated with a higher risk of persistence: 93.8% of participants with breathing difficulties versus 62.4% of participants without breathing difficulties confirmed having at least one persistent symptom (P 5 0.012, OR 5 9.034, 95% CI: 1.160-70.368,data not shown).The logistic regression model suggests that breathing difficulties during the infection and female sex are predictors of symptoms' persistence (P 5 0.033, OR 5 9.489, 95% CI: 1.195-75.297and P 5 0.009, OR 5 2.670, 95% CI: 1.275-5.589,respectively), corroborating the results of the univariate analyses.

DISCUSSION
Here we report the results of an online questionnaire applied to symptomatic dengue patients in Madeira Island, with clinical diagnosis in 2012-2013, at the time of the single dengue outbreak in this population.According to the records of healthcare services, seeking medical assistance primarily occurred during the febrile period of dengue infection, leading to an underestimated clinical picture of the disease because some dengue manifestations only appear later.Additionally, because most of the patients were not followed up, no registry of expanded manifestations exists beyond the acute phase of the infection.Our questionnaire aimed to characterize symptoms retrospectively during active infection and to assess health status before and after dengue diagnosis, identifying long-term manifestations that may be triggered by dengue.As mentioned earlier, we expected self-reported manifestations to be a good representation of those verified at first exposure to the DENV1 serotype that were not biased by immune responses mounted against previous DENV infections.Our results suggest that symptom persistence after primary dengue might be more frequent and longer lasting than anticipated, with a potential impact on the health status and well-being of the affected patients.
The most frequent symptoms during the clinical period were fever, myalgia, extreme tiredness, and headaches, reported by more than three-quarters of our participants, whereas vomiting, pruritus, nausea, retro-orbital pain, and arthralgia occurred in 50% to 35% of participants.In a subset (n 5 67) of this same infected population, Freitas et al. 23 described similar clinical manifestations as the most common.However, their frequencies cannot be directly compared with our study because they referred only to hospitalized patients and to the most relevant symptoms motivating their search for medical  care and/or whose onset occurred during hospitalization.Conversely, our study included nonhospitalized participants and retrospectively analyzed the entire period of symptoms, from the early-acute to the convalescence phase.][12][13][24][25][26][27][28][29] It is noteworthy, however, that gastrointestinal symptoms were 1.5-to 2.7-fold less frequent in our study compared with others. 7,8,10,12,13,24,25,28Hemorrhagic manifestations, when considered altogether, were mentioned by our participants at similar or slightly lower proportions than in other studies. 6,8,29However, marked differences between studies are found when hemorrhagic signs are analyzed individually.For example, gum bleeding and epistaxis were 1.6-to 4.2-fold less frequent in our study, whereas petechiae and hematuria were 1.4-to 5-fold more frequent in our study than in previous research. 6,7,28,29In 2015 and 2020, primary DENV1 local transmission in nonendemic European regions occurred in France 30 and Italy, 31 respectively, and both described similar clinical outcomes to our study.Nevertheless, direct comparisons to our study are impossible because of their small number of cases (N #12).In Effler et al., 29 which included a naive population in Hawaii facing a DENV1 outbreak, most symptoms were 1.4-to 2.2-fold more frequent than in our study, including hemorrhagic symptoms (except for petechiae, which were found at slightly lower proportions).We recognize that there might be an overestimation of petechiae in our study (32.7%) because, from our previous experience with a face-to-face questionnaire, participants tend to misinterpret these as other nonhemorrhagic cutaneous manifestations, and thus their frequency should be interpreted with caution.
Gastrointestinal and hemorrhagic manifestations are considered warning signs and predictors of severity, more commonly present in secondary infections, and known to be influenced by DENV serotype/genotype (reviewed in Yuan et al.). 32Nevertheless, mild hemorrhagic manifestations have been observed in uncomplicated dengue, including in primary infections. 29,33e results of different studies, including those presented here, suggest therefore that hemorrhagic manifestations in primary infections may be more common than anticipated.As for differences between studies addressing symptoms during the infection, we must consider that most studies were held in endemic areas and do not discriminate between primary and secondary dengue, whereas our data are compatible with an expected milder clinical outcome in primary infections.Furthermore, their different designs, including age and sex distribution, forms of assessment and duration of follow-up, and differences in DENV serotypes and host population backgrounds, with distinct genetics and immunological experiences (introduced by natural exposure and vaccination plans), hinder direct comparison.
Most of our participants (72.6%) reported not having any chronic diseases before dengue, what is concurrent with the findings of Martelli et al., 8 where 85.4% of the participants perceived their general health status before the infection as being very good or good, but discordant with Teixeira et al., 13 in which 55.8% of the participants had previous comorbidities.The most frequently reported chronic diseases in our cohort are also not surprising because these are among the most prevalent in our population background. 34When investigating possible correlations between previous chronic disease and each dengue symptom, hypertension was found to correlate significantly with an increased risk for arm and leg swelling but was protective against retro-orbital pain.Apart from the influence of hypertension in the progression of the viral infection to severe forms of dengue, 35 likely potentiated by the pro-inflammatory state of hypertense patients, 36 peripheral edema can also be explained by the condition itself, 37 and/or side effects of antihypertensive drugs. 38We here also hypothesize about the influence of antihypertensive drugs in reducing retro-orbital pain in dengue patients under these therapeutics because several of these drugs have been shown to reduce headaches, given their effects on vascular tone in the cerebrovascular circulation. 39ersistence of clinical manifestations after dengue has been previously reported and may be caused by systemic damage during the acute phase and/or host-related factors affecting their recovery.12][13][14]24 In our study, 61.5% of the participants reported at least one persistent symptom in the 8 years after dengue, of which 68.1% sought medical assistance, thus representing a measure of intensity/persistence of their symptoms.Most of the existing literature describes similar persistent symptoms to those reported in our study, although generally at much lower frequencies.12][13]24 In some of these studies, abundant hair loss ranged from 2.1% to 19.5%, 6,10,13,24 whereas it reached 30.8% in this study.Also, in previous studies, persistent arthralgia and retroorbital pain represented, respectively, 4.5-11.5%, 6,10,13,24and 1.1-3.0%, 10,11,24whereas these were described by 25.7% and 9.6% of our participants.
Previous studies also indicate a resolution of symptoms in most patients within a few months after infection (persistence rates of 8.5-33.3% at 1-2 months 10-12,24 and 7.6-11.5% at 6 months 6,13 ), ending their follow-up after these time windows.As mentioned before, discrepancies between studies can be due to myriad factors, such as differences in study design (age and sex distribution, forms of assessment, and time of follow-up), but also due to biological effects, such as differences in virulence, viral load, and tissue tropism between DENV serotypes, and host comorbidities, immunological status, and genetic susceptibility.Furthermore, the perception of health status may be variable in different populational and cultural backgrounds.
To our knowledge, the only study reporting long-term persistent symptoms is that of Garc ıa et al., 14 in which 56.7% of Cuban participants had at least one chronic symptom 2 years after dengue.Their findings are similar to ours, except for differences noted for persistent headaches, abundant hair loss (respectively, 2.4-and 3.3-fold less frequent than in our study), and retroorbital pain (1.5-fold more frequent than in our study).We should stress that the Garc ıa et al. study 14 was undertaken in a dengue-endemic area, whereas ours refers to primary dengue.In this context, our results suggest that, even in naive populations, the occurrence of persistent symptoms may be more frequent than previously thought and such symptoms may prevail for several years.Nearly 20% of our participants have described persistent ophthalmological signs such as blurred vision and/or retroorbital pains in the years after dengue, four of which have developed ophthalmologic chronic disease.There is, in fact, an increasing body of evidence about dengue-associated eye disease, the onset of which has been described to occur immediately or a few months after the infection (reviewed in Ng and Teoh 2015). 40lso noteworthy, our data are, to our knowledge, the first to investigate and suggest sex and age as good predictors of specific symptoms, either during the infection period or persistent symptoms.During the infection, pruritus, vomiting, nausea, extreme tiredness, palmar erythema, petechiae, and arm and leg swelling were significantly more frequent in females, and arm and leg swelling were more common in older participants.The effect of these variables in an active infection is likely due to hormonal and psychosomatic differences and/or the co-occurrence of age-related conditions.
As for persistent symptoms, headaches, and abundant hair loss were more common in females, whereas headaches were more frequent in younger participants, and arthralgia and bone pain in older participants.Although there is no consensus about the physiopathological mechanisms underlying the persistence of symptoms, it has been suggested that the excessive cytokine production during the acute phase 41 may cause neuroendocrine, musculoskeletal, and immunological damage, 42 which may affect males and females in different ways and lead to more durable consequences in older patients, given their lower regenerative capacity of affected tissues.
Nevertheless, the results presented here should be approached with caution due to a few limitations of our study.All participants had clinical diagnosis of dengue according to the WHO, 4 following the recommendations of ECDC to the regional healthcare authorities, but given the anonymous nature of the questionnaire, it is not possible to estimate the proportion of participants with laboratory-confirmed dengue.Therefore, we must consider that our results may be biased by misdiagnosed cases, caused by other infectious or noninfectious health conditions.Nevertheless, an ongoing study by our team in a subset (n 5 119) of invited participants showed that 8 years after the outbreak, more than 90% were positive for ELISA anti-DENV IgG and foci reduction neutralization test (Henriques et al., submitted for publication).This is in accordance with publications in nonendemic areas, reporting a loss of seropositivity in adults, of approximately 4% per 3-year period 43,44 ; and indicates that most of our participants were, indeed, true dengue cases.Also, we must consider the subjectivity of self-reported data, possible misinterpretation of the questions/multiple answers, and a higher representation of participants with more severe complaints.Moreover, data reports to a reference period from 2012 to 2021, which introduces recall bias.Lastly, we recognize that a sex-and agematched, noninfected, asymptomatic, or population-based control sample would have been relevant for distinguishing persistent symptoms resulting from dengue from those naturally occurring in the population.However, because our study was undertaken several years after the outbreak, we found it unlikely, and prone to recall bias, that a sample set not affected by symptomatic dengue could identify recurrent previously nonexistent symptoms.
In sum, despite its limitations, this study's findings contribute to the existing body of knowledge with a description of the symptoms during primary dengue in a naive population where a single DENV1 outbreak occurred.Our study also illustrates that even in a primary infection, dengue may have significant implications on the health status and well-being of a considerable proportion of the infected population because recurrent symptoms may persist for several years.These data can therefore be considered in terms of healthcare policies, guiding new procedures and strategies for disease control and prevention, and strengthening the importance of the clinical follow-up of dengue patients.Disclosure: The study protocol was approved by the Ethics Committee for Health of the regional healthcare services of "Servic¸o de Sa ude da Região Aut onoma da Madeira (RAM), Entidade P ublica Empresarial da RAM" (approval no.49/2021).
This is an open-access article distributed under the terms of the Creative Commons Attribution (CC-BY) License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

FIGURE 3 .
FIGURE 3. Frequency of participants reporting different symptoms at the time of dengue (overall and per sex).The "Others" category includes loss of appetite, weight loss, ageusia, anosmia, loss of visual acuity, chest pain, and loss of consciousness.Pearson x 2 or Fisher's exact tests were used, with Fisher's P-values (*) shown when at least one of the expected values was ,5; odds ratios (ORs) and 95% CIs are indicated only for variables found to be significant (in bold); n.d. 5 not determined.

TABLE 1
Mean age of participants reporting or not reporting each symptom at the time of dengue infection (overall and per sex) n.d. 5 not determined.The "Others" category includes loss of appetite, weight loss, ageusia, anosmia, loss of visual acuity, chest pain, and loss of consciousness.t-test or Mann-Whitney (*) tests were used according to the normality of distribution accessed by Shapiro-Wilk tests.Significant P-values are highlighted in bold.†To compare mean age of participants with and without symptoms.

TABLE 3
Frequency and mean age of participants reporting recurrent symptoms after dengue (overall and per sex)